Final Report Summary - DIFFEBIMG (Differentiation dynamics in size-controlled embryoid bodies) Project objectives:Aim 1 - Develop an imaging system for size-controlled embryoid bodies (EBs): Design microfluidic chips for controlling the dimensions of forming EBs; Develop a fluorescent marker system for determining the cardiomyocyte fate and intermediate cell states.Aim 2 - Decipher cardiac differentiation in EB development in “wild type” conditions: Image size controlled EBs in “wild type” conditions. Analyze the conditions for appearance of cardiomyocytes: supporting cell types, signaling events, spatial location.Aim 3 - Validate differentiation rules found in Aim 2 and improve cardiac differentiation protocol: Image and analyze EB development while inhibiting/inducing specific signaling pathways. Develop and test improved protocols for cardiac cell differentiation.Description of the work performed:We have developed two types of growth assays for embryoid bodies: 1) microfluidic devices and 2) microwell arrays. In microfluidic devices, we have designed and tested several device types. These devices all contain an array of U-shaped traps, in which embryoid bodies can cluster and form. The designs differ in the geometry of the traps, whether they are one or two-layered, allowing for medium flow above the trap, and in the number and geometry of notches, allowing for flow through the trap. A variant of this device was used in an ongoing collaboration to image the interactions of B cell - T cell pairs. We plan on using this variant in the future for monitoring interactions between isolated pairs of cells.In microwell arrays, we have successfully designed and manufactured arrays of wells from agar or PDMS ranging from 200 micron to 500 micron in diameter. We have successfully established a protocol for forming hundreds of uniform-size EBs within an array of microwells. We have settled on a differentiation protocol that results in high fraction of beating EBs, containing patches of cardiomyocite cells. The EBs are grown, differentiated and imaged within the microwells for a period of two weeks and more. We have established a two-photon laser scanning microscopy system in our lab, for the purpose of live imaging the developmental dynamics of EBs, with emphasis on the relation between signaling events and differentiation decisions. This system was purchased after extensive tests we have made on different confocal microscopy systems. These tests have shown that a two-photon system is most suitable to our task in terms of its penetration power into dense tissue such as EBs.In the last two years we have established a number of fluorescent embryonic stem cell lines with markers for specific developmental decisions. These cell lines are used to assemble EBs, enabling the live monitoring of the relevant developmental stage. The marker lines include pluripotency markers (Nanog-GFP), early mesoderm (Brachyury-GFP) and cardiac progenitor (NKX2.5-GFP). Some of these lines were supplemented with a constitutive fluorescent cell-tagging markers (such as H2B-strawberry). We have recently moved to a gene tagging approach based on the CRISPR technology, with promising results. We are now in the process of evaluating marker lines for several Wnt and BMP pathway signaling molecules and target promoters.We have extensively imaged EBs under default differentiation conditions, monitoring in them mesodermal development (through Bry-GFP) as well as cardiac progenitor stage (Nkx2.5-GFP). We have analyzed the resulting 3D image series through our combined pipeline of custom written code and commercial software. On the intervention side, we collaborated with a lab that mastered a technique for creating a single signaling focus within an EB using fluorescent beads coated with covalently-bound signaling molecules (specifically Bmp4). We have conducted several live imaging experiments to monitor the effect of such focal signaling on early mesodermal differentiation combining their technique with EB imaging system.To summarize, our combined system for wild type and interventional monitoring and analysis of spatio temporal developmental changes in 3D tissue, and their dependence on signaling events is promising to be a strong and unique tool. We expect to use this tool in the years to come to elucidate connections between signal and cell fate.